Multiplex PCR electrospray-ionization mass spectrometry (ESI-MS): Application to forensic mitochondrial DNA examinations

Mitochondrial DNA (mtDNA) typing now can be achieved using electrospray-ionization mass spectrometry (ESI-MS). This method calculates the base composition profile for a DNA fragment by measuring the molecular masses of the light and heavy strands of a PCR product. Differences in base composition are correlated to genetic differences among individuals. The ESI-MS method described by Hall et al. [] is robust, reliable, and automatable, and achieves approximately 85% of the resolving power of sequencing. Thus, base composition analysis is one of the most informative mtDNA SNP-based analytical methods developed to date.

Because base composition analysis does not provide discreet base location, some substitutions can balance one another and mask genetic information. For example, within a 100 base fragment two individuals (1 and 2) differ only at two positions—np 24 and np 83. Sequencing would reveal that these two individuals have different mtDNA types. However, if at position 24, individual 1 had an A (which resulted from a G to A transition) and at position 83 individual 1 had a G (which resulted from an A to G transition), individuals 1 and 2 would have the same base composition and could not be differentiated. With longer fragments there is a greater chance for two balancing substitutions to occur, reducing the resolving power per comparison. In the original procedure described by Hall et al. [], amplicons were restriction digested to reduce the fragment sizes to less than 140 nucleotides in length, a requirement for base composition analysis. The next generation of this ESI-MS method eliminates the restriction digestion and increases the number of amplicons to 24 overlapping PCR fragments, generated in 8 triplex reactions, covering nucleotide positions 15,924–16,428 and 31–576. Amplified fragments range in size from 85 to 140 nucleotides and all triplex reactions amplify under identical reaction conditions. The greater number of small-sized fragments increases coverage and reduces the chance of two balancing substitutions within the same amplicon; this ESI-MS method achieves approximately 94% of the resolving power compared with sequencing. Amplification of small-sized amplicons has the added benefit of increased sensitivity and better success for analyzing degraded DNA.

Fig. 1 represents part of the data output from a base composition analysis. The coverage map is recreated to display all 24 amplicons (PCR products as bars) and their position in the mtDNA genome. The mass measurement error for the product is expressed in parts per million (ppm). Calculated base compositions for each double-stranded product are listed with corresponding nucleotide positions. The images on the right are spectral schematic views for each of the triplex PCR reactions. Each reaction is color coded with the corresponding primer pair indicated at the top of the panel.

This ESI-MS method consistently yields reliable results for mtDNA derived from buccal swabs, blood stains, telogen hairs, bones, and teeth. Additionally, heteroplasmic samples (both point/sequence and length) and mixtures of different known mtDNA types can be quantified and deconvolved, provided there are no balancing substitutions with in an amplicon. Base composition analysis by ESI-MS is a rapid, robust, reproducible, precise, and sensitive method capable of capturing individual-specific variation and resolving mixtures of mtDNA types.

This is publication number 07-08 of the Laboratory Division of the Federal Bureau of Investigation. Names of commercial manufacturers are provided for identification only, and inclusion does not imply endorsement by the Federal Bureau of Investigation.